Method for extraction of Chinese herbal medicine using small molecule micro-shear technology

ABSTRACT

A method for extraction of Chinese herbal medicines using a small molecule micro-shear technology, including: pretreating, jet milling, grinding, pulverizing, cell-wall breaking, and extraction of Chinese herbal medicines, so that the raw material can be fully utilized. Herbs are cut into small molecule substances, which is easy to be absorbed by the means of a superfine grinding technology and a cell-wall breaking technology. By means of a supercritical carbon dioxide extraction technology, active ingredients in Chinese herbal medicines can be efficiently and singly extracted, and organic solvent residues in the raw herbs can be removed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 application of International PCT applicationserial no. PCT/CN2016/080467, filed on Apr. 28, 2016, which claims thepriority benefit of Chinese application no. 201610190063.3, filed onMar. 30, 2016. The entirety of each of the abovementioned patentapplications is hereby incorporated by reference herein and made a partof this specification.

BACKGROUND Technical Field

The invention relates to the field of drug extraction, particularlyrelates to a method for extraction of Chinese herbal medicines usingsmall molecule micro-shear technology.

Description of Related Art

The development of pharmaceutics has experienced four stages: the firstgeneration of preparations is general dosage form, such as capsules,tablets, suppositories, injections, etc.; the second is prodrugs andsustained and controlled release preparations; the third is controlledrelease preparations; and the fourth is targeted preparations. Thedevelopment of new pharmaceutics, technologies, processes and excipientsare inseparable from the four stages of pharmaceutics development.

At present, the pharmaceutical delivery system, namely the newpharmaceutics of third and the fourth generation, has become animportant development direction in the field of pharmacy. The sustainedand controlled release drug delivery system, transdermal drug deliverysystem and targeting delivery system are the mainstream of developmentand hot spot of research. The specific directions are as follows: thesustained and controlled release drug delivery system: the sustained andcontrolled release drug delivery system, also known as the sustained andcontrolled release preparations, which is the fastest growing new drugdelivery system. The drug is made into a sustained and controlledrelease drug delivery system by using sustained and controlled releasepreparation technology, which can release at a pre-designed rate, anddeliver into the body safely and effectively. Comparing to thetraditional preparations, the sustained and controlled release drugdelivery system can reduce the times of doses and improve the complianceof patients; the sustained and controlled release drug delivery systemcan also reduce “peaks and valleys” fluctuations in blood concentrationand toxic side effects, and improve the efficacy; and it can alsoincrease the stability of the treatment, avoid the irritating effects ofcertain drugs on the gastrointestinal tract. According to theadministration route, the sustained and controlled release preparationshave various forms, such as oral sustained and controlled releasepreparations, injection sustained and controlled release preparations,and implantable sustained-release preparation, etc.

Transdermal drug delivery system: the transdermal drug delivery systemrefers to a controlled release preparation, which can rapidly penetratethe skin and enter the blood circulation to act as a systemic treatmentafter transdermal administration. The transdermal drug delivery systemhas the following unique advantages over the general administrationmethods, the transdermal drug delivery system can eliminate the “firstpass effect” of the liver and the destruction of the gastrointestinaltract, and can be stored in the skin layer, which makes the drugconcentration curve more gently, avoiding the “peak valley” andproviding a predetermined and long duration of action. The transdermaldrug delivery system also can maintain a stable and long-lasting bloodconcentration, has low toxicity and adverse reactions, and is convenientto use.

Targeting delivery system: using the liposome, nanoparticle,microspheres and other microparticle carriers, ligands or antibodymicroparticles to target therapeutic drugs to the lesion location, whichhas little effect on non-target tissues, organs and cells, therebyimproving efficacy and reducing the side effects of the drug.

The utilization rate of active ingredients in Chinese herbal medicineshas always been a key issue that restricts the efficacy of the aboveadministration routes. The graininess of the Chinese herbal medicinesgreatly affects the release and the subsequent extraction of the activeingredients in the drugs. Traditional Chinese medicine decoction pieceshave a history of application for thousands of years, but they havedefects such as inconvenient usage, lagging quality standards, anduneven quality. Scholars from countries and regions with moretraditional Chinese medicines such as China, Japan, South Korea andTaiwan have done a lot of exploration and research work on thedevelopment of Chinese herbal medicines. Up to now, traditional Chinesemedicine decoction pieces have experienced four generations,respectively, traditional Chinese medicine decoction pieces (Chinesemedicine decoction pieces collected in “Chinese Pharmacopoeia” or localstandards), granular decoction pieces (Japan or domestic andinternational industrial feeding pieces), single Taste extract granules(Chinese formula granules) and Chinese medicine broken wall pieces(Super Micro Pieces or Taiwanese single-flavored Chinese medicinepowder). Traditional Chinese medicine decoction pieces (currentPharmacopoeia or local standards) is one of the main methods of clinicalapplication of TCM, which has thousands of years of application andtheoretical understanding. It can add or subtract medicine according tothe disease, and it needs to be boiled when used. However, it is easy tohave problems of uneven and uncontrollable quality due to the roughnessand difference in the form of the medicine, and the method of boilingand taking is cumbersome. The traditional long-time and high-temperatureboiling is easy to damage and loss natural active ingredients in thedrug. In this way, the raw materials of Chinese medicinal materials cannot be fully utilized, resulting in a large amount of waste, and it isalso easy to make the dose of the active ingredients in the traditionalChinese medicine preparation unstable.

The efficient extraction of active ingredients is an importantprerequisite for the deep processing of Chinese herbal medicines. Atpresent, the extraction technology of Chinese herbal medicines is mainlybased on traditional methods such as water extraction alcohol extractionand so on. However, heating, organic solvents, etc. destroy activeingredients to a large extent, such as polypeptides and proteins whichare not heat-resistant in Chinese herbal medicines, and the release rateof active substances and the utilization ratio of water-solublesubstances in Chinese medicinal materials are significantly lowered.Therefore, the development of the efficient extraction of activeingredients is of great significance for improving the added value ofChinese herbal medicines.

The principle of the supercritical fluid extraction and fractionationprocess is to utilize the special dissolution of supercritical carbondioxide for certain special natural products to extract and separate.The solvent strength of the supercritical fluid depends on thetemperature and pressure of the extraction. By using this property, itis only necessary to change the pressure and temperature of theextractant fluid, and the different components in the sample can beextracted according to the solubility in the fluid one after another.The weakly polar components are first extracted under low pressure. Asthe pressure increases, the more polar and large molecular weightcomponents are separated from the basic solute.

The Chinese herbal medicines are directly used for extraction afterpreparing into the coarse powder through a simple processing process.Due to the large particles and small specific surface area, the activeingredients cannot be fully extracted, the medicinal materials arewasted, and the active ingredients are not comprehensive. The ultrafinepulverization technique or the nanotechnology are subsequentlydeveloped, which can process the medicines into ultrafine powders. ThePatent Application No. 2009101934934 discloses a method of separatingborneol by using the supercritical CO₂ fluid extraction system. Themethod used CO₂ as a solvent to dissolve the crude borneol, and arelatively pure borneol product can be isolated. Although the ultrafinepulverization technique has made significant progress compared totraditional technology, its essence is still processed by differentphysical means. Since the biologically active substance exists in thecells, simple pulverization, no matter how fine, cannot effectivelyrelease various active ingredients in the Chinese herbal medicines, andthe human body is difficult to effectively absorb and utilize them.Although the physical changes of the particle clusters or moleculargroups in the material are beneficial to the dissolution of the activeingredients, the solubility of the active ingredients is not changed.Therefore, the problem of poor water solubility and low bioavailabilityof some active ingredients has not been fundamentally solved. Therefore,the key to the research of new technology processing technology is toincrease the solubility of active ingredients, enhance the watersolubility of active materials, and meet the requirements of industrialproduction.

SUMMARY

In order to solve the problem of the large particle size and the lowextraction rate of the Chinese herbal medicines, the poor dissolution ofthe active ingredient, the large extracted molecules, and the poorbioavailability caused by the difficulty absorption in the externaladministration mode in the prior art, the present invention provides amethod for extraction of Chinese herbal medicines using small moleculemicro-shear technology.

A specific embodiment of the present invention is to provide a methodfor extraction of Chinese herbal medicines using small moleculemicro-shear technology, and the specific steps of the method are asfollows:

-   -   (1) pretreating raw materials of Chinese herbal medicines, which        requires the moisture content ≤8.0%;    -   (2) initially pulverizing the Chinese herbal medicines until it        can pass through a 200 mesh sieve;    -   (3) pulverizing the pulverized medicinal material by a        supersonic jet mill, and the gas flow pressure is between 0.1        and 0.8 MPa;    -   (4) adding bio-auxiliaries and acidic chemical auxiliaries to        the pulverized powder, grinding them into a slurry, then        filtering and recovering the slurry;    -   (5) treating the mixture obtained in the step (4) by using an        ultra-fine lamination ball mill for 10-15 min;    -   (6) putting the entrainer and the materials treated by the        ultra-fine lamination ball mill into the extraction kettle of        the supercritical fluid extractor, and the supercritical fluid        flow rate into the extraction kettle is 30-40 L/h, the        temperature of the extraction kettle is 40-55° C., and the        pressure is 30-32 MPa, the time of extraction with supercritical        fluid is 6-7 h;    -   (7) taking the fluid from the extraction kettle into the middle        of the separation column to separate the fluid by the first        stage;    -   (8) taking the fluid passed through the separation column into a        stirred first separation kettle to separate it by the second        stage;    -   (9) taking the fluid separated by the first separation kettle        enters the stirred second separation kettle.

Preferably, in the step (3), the gas flow pressure is 0.2-0.5 MPa.

Preferably, in the step (4), the amount of the bio-auxiliaries added tothe pulverized powder is 2-3 wt % of the powder; and more preferably,the bio-auxiliaries is one or a combination of two or more of pepsin,trypsin and maltase.

Preferably, in the step (4), the amount of the acidic chemicalauxiliaries added to the pulverized powder is 1-2 wt % of the powder;and more preferably, the acidic chemical auxiliary is a 65% ethanolsolution of oxalic acid.

Preferably, the separation column has a pressure of 10-15 MPa and atemperature of 60-70° C., and the first separation kettle has a pressureof 5-8 MPa and a temperature of 20-55° C., the second separation kettlehas a pressure of 5-8 MPa and a temperature of 20-55° C.

The advantages of the invention are:

The effects of micro-shear-auxiliary interaction technology andsuper-dispersion pulverization technology on extracting Chinese herbalmedicines are as follows: The organic solvent for extracting was 65%ethanol at the same extraction conditions, and when the solvent waswater, the temperature was set at 64° C. In order to elute the activeingredients sufficiently, the extraction time was selected for 3 hours.The result shows that the extraction effect of the micro-shear-auxiliaryinteraction technology is significantly better than ultra-finepulverization technology. And the selection of active substances andauxiliaries in the micro-shear-auxiliary technology extract is veryimportant, which directly affects the extraction effect of the targets.Proper auxiliaries can significantly increase the yield of the targetand its solubility in the water.

After the cutting and extraction treatment by the method of the presentinvention, the effective ingredient in the drug is sufficiently releasedin the form of small molecules. The drug can be directly applied to theaffected area, directly absorbed by transdermal without any carrier,which can be quickly targeted to the affected area.

The extraction process of the present invention is simple, the operationis convenient, and the extraction time is only 20-30 min. Since theamount of organic solvent is reduced during the extraction process, itis more environmentally friendly.

The method of the present invention can greatly improve the extractionrate of the medicinal material, due to the small molecular size of theextracted medicinal materials, the bioavailability is greatly improved,and therefore, the activity is greatly improved, which has a goodapplication prospect.

DESCRIPTION OF THE EMBODIMENTS Example 1

(1) Pretreating 500 g panax notoginseng raw materials removed of mildewand impurity until the moisture content ≤8.0%.

(2) Initially pulverizing the Chinese herbal medicines material until itcan pass through a 200 mesh sieve.

(3) Pulverizing the pulverized medicinal material by a supersonic jetmill, and the gas flow pressure is between 0.1 and 0.8 MPa.

(4) Adding 2-3 wt % of maltase and 1-2 wt % of 65% ethanol solution ofoxalic acid to the pulverized powder, grinding them into a slurry, thenfiltering and recovering the slurry.

(5) Treating the mixture obtained in the step (4) by using an ultra-finelamination ball mill for 10-15 min.

(6) Putting 120 mL of propylene glycol and the materials treated by theultra-fine lamination ball mill into the extraction kettle of thesupercritical fluid extractor, and the supercritical fluid flow rateinto the extraction kettle is 40 L/h, the temperature of the extractionkettle is 52° C., and the pressure is 32 MPa, the time of extractionwith supercritical fluid is 7 h.

(7) Taking the fluid from the extraction kettle into the middle of theseparation column to separate the fluid by the first stage. Theseparation column has a pressure of 12 MPa and a temperature of 65° C.

(8) Taking the fluid passed through the separation column into a stirredfirst separation kettle to separate it by the second stage. The firstseparation kettle has a pressure of 5.5 MPa and a temperature of 55° C.

(9) Taking the fluid separated by the first separation kettle enters thestirred second separation kettle. The second separation kettle has apressure of 5 MPa and a temperature of 45° C.

Example 2

(1) Pretreating 500 g pinellia ternate raw materials removed of mildewand impurity until the moisture content ≤8.0%.

(2) Initially pulverizing the Chinese medicinal material until it canpass through a 200 mesh sieve.

(3) Pulverizing the pulverized medicinal material by a supersonic jetmill, and the gas flow pressure is between 0.2 and 0.5 MPa.

(4) Adding 2-3 wt % of trypsin and 1-2 wt % of 65% ethanol solution ofoxalic acid to the pulverized powder, grinding them into a slurry, thenfiltering and recovering the slurry.

(5) Treating the mixture obtained in the step (4) by using an ultra-finelamination ball mill for 10-15 min.

(6) Putting 120 mL of propylene glycol and 250 g materials treated bythe ultra-fine lamination ball mill into the extraction kettle of thesupercritical fluid extractor, and the supercritical fluid flow rateinto the extraction kettle is 40 L/h, the temperature of the extractionkettle is 52° C., and the pressure is 32 MPa, the time of extractionwith supercritical fluid is 7 h.

(7) Taking the fluid from the extraction kettle into the middle of theseparation column to separate the fluid by the first stage. Theseparation column has a pressure of 12 MPa and a temperature of 65° C.

(8) Taking the fluid passed through the separation column into a stirredfirst separation kettle to separate it by the second stage. The firstseparation kettle has a pressure of 5.5 MPa and a temperature of 55° C.

(9) Taking the fluid separated by the first separation kettle enters thestirred second separation kettle. The second separation kettle has apressure of 5 MPa and a temperature of 45° C.

Test Example 1

The forsythia was extracted by the method of Example 1 of the presentinvention and the method of Example 1 of Patent No. 2009101934934(Control), and the therapeutic effects of both on the swelling of theear shell skin of the mouse were examined. The experimental steps are asfollows:

Experimental principle: Using inflammatory agents to cause swelling ofthe ear skin, and the weight of the inflamed ear shell of the appliedmedicine group and the control group was observed and measured, and thedifference in swelling rate was compared.

Operation Method:

(1) Male mice of 25 to 30 g were used.

(2) Under anesthesia of ether, about 0.1 mL the mixed pro-inflammatorysolution was applied to both sides of the left ear of the mouse (Theinflammation solution contains 2% croton oil, 20% pyridine, 5% distilledwater and 73% diethyl ether). The right ear is used as a control.

(3) 0.03 mL/kg of test drug was injected intraperitoneally 0.5-1 hourbefore the inflammation.

(4) After 4 h, the mice were sacrificed, and the ears were cut. Theoriginal ear pieces were taken off with a 9 mm diameter puncher on thesame part, and weighed with a balance. The weight of the left ear pieceper mouse minus the weight of the right ear piece is the degree ofswelling. The mean value and standard deviation of the swelling degreeof each group were calculated, and the swelling inhibition rate of theadministration group was determined according to the formula. Theformula is: swelling rate a=(inflamed ear weight−uninflamed earweight)/uninflamed ear weight.

(5) Both the applied drug and the control group were diluted to 1%.

The experimental results are as follows:

Number of Swelling Drug animals rate Example 1 20 0.67 ± 0.05 Example 218 0.73 ± 0.09 Control group 18 1.62 ± 0.12

From the above results, it was found that the swelling rates of themouse ear pieces prepared by the method of the present invention wereall lower than that of the control group. The pulverization andextraction method of the present invention allows the effectiveingredients in the Chinese herbal medicines to be completely retainedand fully utilized, so that the obtained drug has a remarkable effect.

The detailed description of the present invention is intended to beillustrative of the preferred embodiments of the present invention, andis not intended to limit the scope of the present invention. Equivalentimplementation or modification without departing from the inventionshall be included in the scope of the technical scheme of the invention.

What is claimed is:
 1. A method for extraction of Chinese herbalmedicines using small molecule micro-shear technology, comprising: (1)pretreating 500 g forsythia raw materials removed of mildew and impurityuntil the moisture content ≤8.0%; (2) initially pulverizing the Chineseherbal medicinal material until it can pass through a 200 mesh sieve;(3) pulverizing the pulverized medicinal material by a supersonic jetmill, and a gas flow pressure is between 0.1 and 0.8 MPa; (4) adding 2-3wt % of maltase and 1-2 wt % of 65% ethanol solution of oxalic acid to apulverized powder, grinding them into a slurry, then filtering andrecovering the slurry; (5) treating a mixture obtained in the step (4)by using an ultra-fine lamination ball mill for 10-15 min; (6) putting120 mL of propylene glycol and materials treated by the ultra-finelamination ball mill into an extraction kettle of a supercritical fluidextractor, and the supercritical fluid flow rate into the extractionkettle is 40 L/h, the temperature of the extraction kettle is 52° C.,and the pressure is 32 MPa, the time of extraction with supercriticalfluid is 7 h; (7) taking the fluid from the extraction kettle intomiddle of a separation column to separate the fluid by a first stage,wherein the separation column has a pressure of 12 MPa and a temperatureof 65° C.; (8) taking the fluid passed through the separation columninto a stirred first separation kettle to separate it by a second stage,wherein the first separation kettle has a pressure of 5.5 MPa and atemperature of 55° C.; and (9) taking the fluid separated by the firstseparation kettle enters a stirred second separation kettle, wherein thesecond separation kettle has a pressure of 5 MPa and a temperature of45° C.
 2. The method for extraction of Chinese herbal medicines usingsmall molecule micro-shear technology of claim 1, wherein the gas flowpressure is 0.2-0.5 MPa.